Iron is an essential but potentially toxic nutrient for virtually all organisms. Iron deficiency is the most common human nutritional deficiency disease. At the same time excessive iron stores have been associated with increased occurrence of neurological disorders and certain cancers. Mammalian iron metabolism is modulated through the action of two regulatory RNA binding proteins, iron regulatory protein 1 (IRP1) and IRP2. IRPs bind to iron responsive elements (IRE) in ferritin (iron storage) and transferrin receptor (TfR) (iron uptake) mRNAs and regulate their translation or stability, respectively. IRP1 is an Fe-S protein and the presence or absence of the Fe-S cluster modulates the RNA binding activity of the protein. Iron regulates the RNA binding activity of a related protein, IRP2, by inducing its degradation. Because IRPs are pivotal regulators of iron metabolism, and directed changes in iron metabolism occur in human health and disease, it is important to better understand how iron and other factors affect IRP function. Our overall goal is to determine how iron metabolism is modulated through the action of intracellular and extracellular effectors that influence IRP action. We have begun to elucidate a novel mechanism by which protein kinase C (PKC)-dependent phosphorylation of IRPs serves as a means through which hormones, growth factors and other agents can act to influence cellular iron metabolism. We propose to: 1) use a structure/function approach to determine how phosphorylation affects IRP function in vitro; 2) determine the cellular role of phosphorylation in the function and compartmentalization of IRPs; and 3) determine the effect of iron and oxidative stress on the cellular function of phosphorylated IRPs. Our broad goal with regard to IRP1 is to understand how regulated changes in assembly and stability of its Fe-S cluster affect its function as an iron-regulated RNA protein. Our overall goal for IRP2 is to delineate how phosphorylation affects the redox and iron-regulation of its proteasomal degradation. Our studies provide a comprehensive approach from the molecular to the whole animal level that will: 1) delineate a novel mechanism for regulated changes in stability of Fe-S clusters in mammals; 2) describe a unique example of how phosphoregulation and iron-regulation overlap to establish the steady state level of the regulatory RNA binding proteins, IRP1 and IRP2; 3) further define the molecular pathways through which mammalian iron metabolism can be regulated.